Structural and regulatory studies on protein components of the E. coli sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate (PEP) on an active site histidine in a Mg(2+)-requiring reaction to produce pyruvate. New studies, in collaboration with the Clore laboratory (NIDDK), have elucidated the three-dimensional structure of the cytoplasmic B domain of the Escherichia coli mannitol transporter using NMR. The ordered IIBMtl domain consists of a four-stranded parallel beta-sheet flanked by two helices on one face and an additional helix on the opposite face with a characteristic Rossman fold. The active site cysteine (Cys-384) is primed for nucleophilic attack at the phosphorylated histidine (His-554) of the IIAMtl domain. The structure of IIBMtl is similar to that of protein tyrosine phosphatase. In collaboration with the Wang laboratory (Eppley Institute), structural analysis of the nitrogen regulatory arm of the PTS has been started. The X-ray structure of IIANtr has previously been reported by others. To lay the groundwork for the deduction of the solution structure of the complex of IIANtr with NPr, the chemical shift assignments for IIANtr have been made. The N-terminal domain of glucose IIA confers amphitropism to the protein, allowing it to shuttle between the membrane and cytoplasm. The structure of a synthetic peptide corresponding to the N-terminal domain was studied. The effects of phospholipids or detergent chain length on the structure and translational diffusion coefficient of the peptide were investigated by NMR. Three anionic phospholipids and four lipid-mimicking anionic detergents were evaluated. In all cases, the cationic peptide adopts an amphipathic helical structure. While the chain-length of the two-chain phospholipids has a negligible effect on the peptide conformation, the effect of chain length of single-chain detergents is more significant. Short-chain anionic phospholipids are proposed to be useful membrane-mimetic models for the structural elucidation of membrane-binding peptides. The bacterial PTS regulates a variety of physiological processes, some of which are mediated by Enzyme IIAGlc. A novel IIAGlc binding protein was discovered by ligand fishing using surface plasmon resonance. The protein, named FrsA (for fermentation/respiration switch protein), is the 47 kDa product of the yafA gene, previously denoted as ?function unknown?. FrsA forms a 1:1 complex with unphosphorylated IIAGlc with a high affinity. Disruption of frsA increased cellular respiration on several sugars, while increased FrsA expression resulted in an increased fermentation rate of some sugars. The results are interpreted to indicate that IIAGlc regulates the flux between respiration and fermentation by sensing the available sugar species via a phosphorylation state dependent interaction with FrsA.